Molecular Mechanism Of Arabidopsis Response To Low Nitrogen And Calcium

Our lab studies molecular mechanism of plants to mineral nutrient deprivation with Arabidopsis as material. This thesis is composed of two relatively independent sections:by screening genetic mutants, we conducted analysis on molecular role of RHD3in low nitrogen-induced anthocyanin accumulation; by combining measuring cytosolic Ca dynamics change in vivo and microarray-based transcriptional analysis, we initiated investigation of cellular and molecular mechanism of plant cell response to low calcium.(1) We isolated a single gene and recessive mutant showing anthocyanin overly accumulation phenotype under low nitrogen, which displayed short primary root, short and wavy root hair, dwarf phenotypes. Combination of positional cloning and next generation sequencing-based DNA polymorphisms analysis identified an single base mutation in the last base of16th intro of ROOT HAIR DEFECTIVE3(RHD3) leading truncated RNA product, which was named as rhd3-10. The mutant’s phenotype was copied by rhd3-1and rhd3-1/rhd3-10F1plants. It demonstrated that RHD3indeed is the gene responsible for all observed phenotype. RHD3locates in ER and its promoter activity primarily is in the leaf and root tip. Mutating TT4, an essential gene for anthocyanin biosynthesis, completely blocked the low nitrogen-induced anthocyanin accumulation in rhd3-10.Under low nitrogen, rhd3-10had higher expression of anthocyanin biosynthesis related genes and accumulation of anthocyanin itself than those of wild type (Col). It demonstrated that RHD3is a negative regulator of the low nitrogen-induced anthocyanin accumulation. Ethylene negatively regulates high light-induced anthocyanin accumulation. We hypothesized that RHD3negatively regulates anthocyanin biosynthesis through modulating ethylene signaling pathway, which was supported by following four evidences:(a) RHD3mutation partially blocked the low nitrogen-induced ethylene responsive gene expression;(b) Under low nitrogen, ethylene signaling blocking mutants etrl、ein2、ein3/eill accumulated more, but ethylene over-producing mutant etol accumulated less anthocyanin. It demonstrated that ethylene indeed is the negative regulator of this process;(c) Ethylene precursor ACC strongly suppressed the low nitrogen-induced anthocyanin accumulation in Col. The suppressive effect of ACC was significantly attenuated in ethylene signaling mutants and rhd3-10;(d) Introducing rhd3-10into etol increased low nitrogen induced anthocyanin accumulation. Though RHD3, ETR1, EIN2and CTR1locate or associate with ER, we didn’t detect protein-protein interaction between RHD3N or C terminals and ETR1, EIN2and CTR1. It implies further investigation needed for exploring molecular mechanism of RHD3-regulated ethylene signaling. (2) Ecological evidence indicates a worldwide trend of dramatically decreased soil Ca2+levels caused by increasing acid deposition and massive timber harvesting. Little is known about the genetic and cellular mechanism of plants’responses to this decrease. In this study, transcriptional profiling analysis identified many extracellular Ca ([Ca2+]ext) deprivation-responsive genes in Arabidopsis thaliana, many of which are involved in plant responses to other environmental stresses. Interestingly, a group of putative cytosolic Ca2+([Ca2+]cyt) sensor-encoding genes were significantly up-regulated, which implied that [Ca2+]cyt has a role in sensing the [Ca2+]ext deprivation. Supportively, decreasing [Ca2+]ext evoked a transient rise in [Ca2+]cyt, the amplitude of which positively correlated with decreasing [Ca2+]ext and negatively [K+]ext.The response was insensitive to an animal Ca sensor antagonist, but was suppressed by neomycin, an inhibitor of phospholipase C. The [Ca2+]cyt response to [Ca2+]ext deprivation was significantly desensitized after the initial treatment, which was typical of a receptor-mediated signaling event. Gd3+, an inhibitor of Ca2+channels, suppressed the [Ca2+]ext triggered [Ca2+]cyt rise and the downstream gene expressions. Taken together, this study demonstrated that [Ca2+]cyt plays an important role in the putative receptor-mediated cellular and transcriptional sensing of [Ca2+]ext deprivation of plant cells.In summary, this study identified a new function of RHD3in plant response to low nitrogen and demonstrated that RHD3negatively regulated low nitrogen-induced anthocyanin biosynthesis through modulating ethylene signaling. On the other hand, this study established the cellular and molecular mechanism of plant response to low calcium. It will pave foundation for conducting further investigation of plant response to mineral nutrition deprivation signals.